The ability to grow premature ovarian follicles in vitro to the advanced antral stage to recover developmentally competent oocytes would be invaluable for enhancing/preserving (1) human fertility and (2) genetically valuable disease models. Live offspring from cultured follicles (and resident oocytes) have been produced only in the mouse. But what works in this species is not directly applicable to women because of significant differences in the duration of folliculogenesis, the size of fully-grown follicles-eggs rigidity of the ovarian extracellular matrix (ECM) and lipid content within oocytes. In short, findings in the mouse have not allowed consistent, competent oocyte recovery in non-rodent models. We propose exploring new and alternative approaches to follicle culture using the cat model that has remarkable similarities in ovarian structure and function to the human. Our objective is to create a physically-flexible and energy-rich microenvironment that consistently produces developmentally competent oocytes from cultured follicles. Others have focused on examining the influences and identifying gonadotropins and growth factors that promote follicle growth in vitro. Although such follicles increase somewhat in size and produce steroids, resident oocyte competence is poor partly because the encompassing ovarian cortex (or culture 'gel') restricts physical expansion of the follicle-oocyte complex. There also has been little attention directed at ensuring adequate energy availability for the growing oocyte. Therefore, our innovative strategy is to circumvent these issues by altering the physical microenvironment to permit follicle expansion while enhancing intracellular lipid as an energy source for the oocyte. We will take two parallel, complimentary approaches, one directed at the ovarian cortex (Specific Aim 1) and the other at isolated ovarian follicles (Specific Aims 2 and 3). In Specific Aim 1 studies, we will artificially remodel the ECM of ovarian tissue by stimulating matrix metalloproteinase (MMP) production that, in turn creates optimal conditions for primordial follicle activation. Specific Aim 2 studies focus on creating a hydrogel network containing MMP-labile peptides that will promote isolated follicle development. Specific Aim 3 studies will target ensuring survival of high quality oocytes enclosed in host follicles by enhancing accumulation of intracellular lipid (via supplementation or de novo biosynthesis). Our plan is bolstered by (1) a multidisciplinary team with decades of experience in bioengineering and gamete/developmental biology and (2) unlimited, free access to cat ovaries from spay/neuter clinics. Our outcome will be a new scholarly understanding of the complex mechanisms regulating folliculogenesis and oocyte development from early and diverse stage follicles, including primordial. Most importantly, our novel approaches and findings will shift the paradigm in how we think about, refine and practice follicle and oocyte rescue. This capacity will access vast amounts of unused ovarian germplasm while offering new fertility preservation options to (1) young women cancer patients and (2) valuable disease models.